For imaging experiments, cells were plated on 35mm glass bottom dishes at low density (MatTek Corp, Ashland, MA). Immunofluorescence and live cell imaging were carried out one day after transfection. Spinning disc confocal (SDC) microscopy was performed using the Improvision UltraVIEW VoX system (Perkin-Elmer) built around a Nikon Ti-E inverted microscope, equipped with PlanApo objectives (40x1.0-NA 1.0 and 60X1.49-NA) and controlled by Volocity (Improvision) software. Excitation light was provided by 488-nm/50-mW diode laser (Coherent) and 561-nm/50-mW diode laser (Cobolt), and fluorescence was detected by EM-CCD camera (C9100–50; Hamamatsu Photonics).
Total internal reflection fluorescence (TIRF) microscopy was performed on a setup built around a Nikon TiE microscope equipped with 60X1.49-NA. Excitation light was provided by 488-nm (for GFP), 561-nm (for mCherry/mRFP/mdsRed) and 640-nm (for iRFP) DPSS lasers coupled to the TIRF illuminator through an optic fiber. The output from the lasers was controlled by an acousto-optic tunable filter and fluorescence was detected with an EM-CCD camera (Andor iXon DU-897). Acquisition was controlled by Andor iQ software. Images were sampled at 0.20 Hz with exposure times in the 100–500 ms range. SDC microscopy was carried out at room temperature (20–25°C) and TIRF microscopy at 37°C.
Total internal reflection fluorescence (TIRF) microscopy was performed on a setup built around a Nikon TiE microscope equipped with 60X1.49-NA. Excitation light was provided by 488-nm (for GFP), 561-nm (for mCherry/mRFP/mdsRed) and 640-nm (for iRFP) DPSS lasers coupled to the TIRF illuminator through an optic fiber. The output from the lasers was controlled by an acousto-optic tunable filter and fluorescence was detected with an EM-CCD camera (Andor iXon DU-897). Acquisition was controlled by Andor iQ software. Images were sampled at 0.20 Hz with exposure times in the 100–500 ms range. SDC microscopy was carried out at room temperature (20–25°C) and TIRF microscopy at 37°C.